基于CH3-HS疏水结构域液相表征特性锚定DNA甲基化特异位点的电化学分析研究

The analysis of locus-specific DNA methylation is the ultimate goal in epigenetic research and clinical detection of DNA methylation. In present, the bisulfite-sequencing technology has been used for the detection of genome methylation, but it has some disadvantages such as the requirements of a deep-sequencing, incomplete conversion and big data analysis. Methylation-sensitive polymerase chain reaction was limited by the endonuclease recognition sequence, and most important of all, the accurate methylation sites could not be identified with this technique. It becomes an imperative requirement to set up an analysis technique for DNA methylation site detection in epigenetic research and clinical laboratory diagnosis..Our previous research showed that the molecular characterization of methylated DNA would change into hydrophobic grouping CH3- in liquidoid, and form a CH3-core hydrophobic sphere. Therefor, in this study, we intend to establish an electrochemical analytic technique for identifying DNA methylation specific site based on the liquidoid characteristic of CH3-hydrophobic sphere structure, the electroneutral backbone pcPNA was used to capture target DNA chain which methylation status is designed at different sites. On the electrode surface, the target CH3 hydrophobic sphere existed the space phase difference in the distribution of the membrane microenvironment. Based on this mechanism, the electrochemical mathematical model was constructed to dectect DNA locus-specific methylation. Then, deeply parse response mechanism of pcPNA electrochemical analysis in liquid phase. With the development, the locus-specific methylation on dsDNA would be studied based on the pcPNA as probe with the characteristic of the double helix invasion. Through this project, we hope to construct a novel original technical of analyzing locus-specific DNA methylation which would support the research of epigenetic and clinical detection of DNA methylation. On the other hand, the related basic theory will be clarified such as the optimum base spacing for electrochemical analysis of DNA methylation.

甲基化特异位点分析是表观遗传DNA甲基化检测的最终目标。目前的基因组Bisulfite-Seq分析存在测序深度、转换效率和庞大数据分析等不足；MSRE分析引物设计受内切酶RE识别序列限制且不能精确定位甲基化位点。本项目在对甲基化DNA分子结构分析和项目组前期研究证实，甲基化DNA液相条件下形成以CH3-为内核的疏水球这一物化特性基础上，利用pcPNA捕获不同位点甲基化的靶序列DNA后，靶序列CH3-疏水球在电极表面膜微环境分布的空间位相差异，建立DNA不同位点甲基化与电化学响应数学模型，达到锚定DNA甲基化特异位点的目的。并在深入解析pcPNA电化学传感液相响应机理基础上，利用pcPNA双螺旋侵袭特性，进一步探索双链dsDNA甲基化位点分析的可行性和基础参数。通过研究，拟为表观遗传研究和临床甲基化检测建立一种原创性的新的技术手段，同时阐明DNA甲基化电化学分析碱基最适间距等相关基础理论。

甲基化特异位点分析是表观遗传DNA甲基化检测的最终目标。目前的基因组Bisulfite-Seq分析存在测序深度、转换效率和庞大数据分析等不足；MSRE分析引物设计受内切酶RE识别序列限制且不能精确定位甲基化位点。本项目在对甲基化DNA分子结构分析和项目组前期研究证实，甲基化DNA液相条件下形成以CH3-为内核的疏水球这一物化特性基础上，利用利用PNA捕获不同位点甲基化的靶序列DNA后，靶序列CH3-疏水球在电极表面膜微环境分布的空间位相差异，建立DNA不同位点甲基化与电化学响应数学模型，达到锚定DNA甲基化特异位点的目的。我们的研究结果显示，距离电极表面越近的甲基化靶序列的峰电流越小，随着甲基化位置逐渐远离电极表面，峰电流逐渐增大。与预期结果相符。并且，我们制备的DNA生物传感器可以达到0.84fM的检测限，具有灵敏度高和特异性好等优点，为DNA甲基化临床检测提供了广阔的应用前景。